Unlocking Nature�s Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter

Rajak, N and Dey, T and Sharma, Y and Bellad, V and Rangarajan, PN (2024) Unlocking Nature�s Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter. In: Microbial Cell Factories, 23 (1).

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Abstract

Background: Komagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (PPEPCK) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins. Results: PPEPCK-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from PPEPCK. The traditional, methanol-inducible alcohol oxidase 1 promoter (PAOX1) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100�120 A600 units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from PPEPCK that is comparable or even higher than that from PAOX1. Conclusions: We have designed an induction medium for recombinant protein production from K. phaffii PPEPCK in shake flask cultivations. It consists of 1.0 yeast extract, 2.0 peptone, 0.17 yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0 MSG, and 2 ethanol. Substitution of ammonium sulphate with 0.5 urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from PPEPCK equaled and even surpassed those from PAOX1. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii PPEPCK in bioreactors. © The Author(s) 2024.

Item Type:	Journal Article
Publication:	Microbial Cell Factories
Publisher:	BioMed Central Ltd
Additional Information:	The copyright for this article belongs to authors.
Keywords:	alcohol; alcohol oxidase; ammonium sulfate; biotin; carbon; formaldehyde; glutamate sodium; glutamic acid; green fluorescent protein; helix loop helix protein; hydrogen peroxide; methanol indudcible alcohol oxidase 1; nitrogen; peptone; phosphoenolpyruvate carboxykinase (GTP); potassium dihydrogen phosphate; recombinant protein; unclassified drug; urea; viral protein; yeast extract; glutamate sodium; glutamic acid; methanol; recombinant protein, Article; bacterial strain; biomass production; carbon source; cell density; enzyme synthesis; gene expression system; gene insertion; Komagataella pastoris; Komagataella phaffii; nonhuman; protein expression; protein purification; protein secretion; receptor binding; Severe acute respiratory syndrome coronavirus 2; substitution reaction; budding yeast; genetics; metabolism; Pichia, Ethanol; Glutamates; Methanol; Pichia; Recombinant Proteins; Saccharomycetales; Sodium Glutamate
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	21 May 2024 04:08
Last Modified:	21 May 2024 04:08
URI:	https://eprints.iisc.ac.in/id/eprint/84600

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